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WO1994012213A1 - Procede de production d'anticorps avec des conjugues de l'antigene de lymphocytes b et une molecule porteuse denaturee - Google Patents

Procede de production d'anticorps avec des conjugues de l'antigene de lymphocytes b et une molecule porteuse denaturee Download PDF

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Publication number
WO1994012213A1
WO1994012213A1 PCT/DK1993/000382 DK9300382W WO9412213A1 WO 1994012213 A1 WO1994012213 A1 WO 1994012213A1 DK 9300382 W DK9300382 W DK 9300382W WO 9412213 A1 WO9412213 A1 WO 9412213A1
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antigen
cell
cell antigen
carrier
immunization
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Ole Buchardt
Claus Koch
Peter Eigil Nielsen
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6081—Albumin; Keyhole limpet haemocyanin [KLH]

Definitions

  • the present invention concerns a process for providing antibodies to haptens and other B cell antigens as well as antibodies obtained by the process.
  • the invention moreover concerns the use of the produced antibodies for vaccines, in particular for veterinary medicinal use.
  • the general immune response is based on a cooperation be ⁇ tween antigen presenting cells, antigen specific T lympho ⁇ cytes and antigen specific B lymphocytes.
  • the T lympho ⁇ cytes are selected and stimulated by T cell antigens which, associated with MHC class II (Major Histocompati- bility Complex) molecules, are presented on the surface of so-called antigen presenting cells; see figure 1.
  • MHC class II Major Histocompati- bility Complex
  • a T cell antigen (a T cell epitope) can be described by the following characteristics:
  • the B lymphocytes are selected by the administered antigen which binds to the antigen specific receptor (membrane bound antibody) of the B cell. Stimulation of the selected B cell takes place by internalizing and processing the antigen, following which the T cell epitope of the antigen is presented on the surface of the B cell, associated with MHC class II molecules. This presentation entails that the selected and stimulated T accessory cells are associated with the antigen specific B lymphocyte, which is now sti ⁇ mulated and matured via hormone-like factors from the T cell (e.g. interleukins) . The final stage of the B cell after stimulation and maturing is the antibody producing plasma cell which secretes antigen specific antibody; see figure 2.
  • B cell antigen (a B cell epitope) can be described by the following characteristics:
  • Substantially any molecular configuration can act as a B cell antigen (epitope). As regards proteins, these epitopes are most frequently positioned on the outer, accessible face of the protein.
  • a B cell antigen can bind directly to the antigen specific receptor (the antibody) of the B cell.
  • Immune response in the form of antibody formation to a given antigen is therefore conditional upon the presence of both B cell epitope(s) and T cell epitope(s). These two types of epitopes must be physically associated.
  • the basis of this knowledge are classic experiments from around 1970 concerning the hapten carrier molecule problems (see K. Rajewsky et al., J. Exp. Med. 129, 1131 (1969), N.A. Mitchison, Eur. J. Immunol. 3 ⁇ , 10 (1971) and N.A. Mitchi- son, ibid. 1, 18 (1971)).
  • Antibody response to low molecular antigen molecules can be induced in a mammal only if the hapten pre ⁇ sented for the animal in question is coupled on an immuno- genie carrier.
  • Another phenomenon which frequently consti ⁇ tutes a problem is that certain molecules, which otherwise have a relatively large molecular size, are only very little immunogenic. This may apply to e.g. larger pep- tides, low immunogenic proteins, carbohydrates, lipids, nucleic acids, etc.
  • carrier molecules useful for inducing or en ⁇ hancing the immune response to such molecules are serum albumin (BSA, HSA and others), keyhole limpet hemocyanin (KLH), ovalbumin (OA) , chicken im unoglobulin (Chlg) and diphtheria toxoid (DT), but a large number of others might be mentioned.
  • BSA serum albumin
  • KLH keyhole limpet hemocyanin
  • OA ovalbumin
  • Cho chicken im unoglobulin
  • DT diphtheria toxoid
  • an immunogenic protein antigen which induces both T cell and B cell immunity under normal circumstances, can be converted to or act as an immunogenic, "pure" T cell antigen to which no or only very little B cell immunity is induced. Then, the molecule to which antibodies are to be produced, can be coupled chemically to this T cell antigen or to a corresponding, modified T cell antigen.
  • T cell immunity can be induced as a general principle in an animal. This can be done while very limited B cell immunity or none at all is induced in relation to the carrier molecule which is subsequently used for induction of the relevant antibody response to hapten or the like.
  • Immune response in the form of antibody formation to the T cell antigen (carrier molecule) can be "opti- mized" to be very low (possibly not measurable).
  • the invention concerns a process for providing antibodies to haptens, peptides/proteins, carbo ⁇ hydrates, lipids, nucleic acids, weak immunogens and other B cell antigens, including combinations of these, in ani ⁇ mals, wherein the B cell antigen introduced in the animal is coupled to an immunogenic carrier molecule in the form of a protein antigen, which normally induces T cell immu ⁇ nity as well as B cell immunity.
  • the process of the inven- tion is characterized by modifying the immunogenic protein antigen to an exclusive or predominant T cell antigen by a denaturating modification of the antigen, coupling the B cell antigen to which antibodies are to be produced to the modified T cell antigen, followed by immunization, optio- nally using an adjuvant as an immune stimulating means, preferably after preim unization with T cell antigen in a form which may be different from the form used in the immunization proper with the B cell antigen.
  • the invention further concerns antibodies or antisera obtained by the present process.
  • the invention concerns use of the antigens produced by the process for the production of vaccines, in particular for veterinary medicinal use.
  • Processes which are related to the process according to the invention for providing antibodies to B cell antigens in animals are known from EP published specification No. 0429816 and from Science 249, 423-425 (1990).
  • These literature sources concern a structure which represents a T accessory epitope and which contains information for a carrier function, but not for a B epitope suppressive function.
  • These T cell structures may stem from the same protein, in which case it will be necessary to remove sequences which contain information for the suppressive function, or alternatively to change these sequences in such a way that they no longer exhibit suppressive function.
  • the T cell epitope may be a sub-part of various structures, especially parts or microorganisms, in particular sequences from tetanus toxoid. It is stated nowhere that the T cell antigen may be denatured whole anti
  • WO published specification No. 89/06974 concerns T cell epitopes from bacterial products and describes how a T cell epitope is defined, isolated, produced and coupled to a B cell epitope.
  • This procedure involves modification of a protein antigen, e.g. in the form of a so-called denaturation of the anti ⁇ gen. If the modification is effected as a denaturation, this may take place by common methods for achieving dena- turation, but preferably by methods comprising blocking charged groups (either -NH heap or -COO ), e.g. for -NH heap by means of formaldehyde, and reducing disulfide bridges, e.g. by means of dithiothreitol or ⁇ 3-mercapto ethanol. Then the actual denaturation takes place by e.g. heat treatment. The T cell antigen then occurs in two forms: a non-modified one and a modified one.
  • This coupling takes place e.g. in one of the following manners:
  • the hapten is modified chemically, so that e.g. a carboxylic, a sulfone or other acid group is converted to an active derivative, e.g. an active ester, an acid halo- genide or an acid anhydride, symmetrically or mixed, which then reacts with the carrier protein with covalent coup ⁇ ling.
  • active derivative e.g. an active ester, an acid halo- genide or an acid anhydride, symmetrically or mixed
  • Other cases may involve activating a mercapto group by converting it to an activated disulfide, which is then caused to react with the mercapto group on the carrier protein (see the following) .
  • the native hapten does not contain a group which can be converted to an activated derivative. This may be handled by modifying the hapten, e.g. by in ⁇ troducing a carboxyl group or mercapto group which can then be activated by introducing ligands with an ⁇ , ⁇ - activated double bond, such as e.g. vinyl sulfonyl, or by introducing a photochemically activatable group, such as e.g. 4-azidobenzoyl or 2- or 3-diazocyclopentadienyl car- bonyl; these couplings may also be established via Hg-S bonds.
  • ligands with an ⁇ , ⁇ - activated double bond such as e.g. vinyl sulfonyl
  • a photochemically activatable group such as e.g. 4-azidobenzoyl or 2- or 3-diazocyclopentadienyl car- bonyl; these couplings may also be established via Hg
  • the carrier protein is not equipped with the neces- sary functional groups, such as e.g. SH, these groups may be introduced chemically before the coupling step takes place.
  • Carrier proteins can be modified so that their func- tional groups are activated, e.g. by converting carboxylic acid groups to active esters or by activating mercapto groups so that these can be coupled to the functional groups of the hapten.
  • Hapten and carrier protein are caused to react with homo- or heterobifunctional groups, such as e.g. divinyl sulfone, dicarboxylic acids, formaldehyde, glutaraldehyde or glutaconaldehyde, active esters of dicarboxylic acids, maleimide active esters, 4-azidobenzoyl derivatives of active esters, etc.
  • homo- or heterobifunctional groups such as e.g. divinyl sulfone, dicarboxylic acids, formaldehyde, glutaraldehyde or glutaconaldehyde, active esters of dicarboxylic acids, maleimide active esters, 4-azidobenzoyl derivatives of active esters, etc.
  • Immunization may comprise preim unization with T cell antigen in a form different from the form which is used in the actual immunization with the B cell antigen. This is followed by the actual immunization with the B cell anti ⁇ gen coupled on a relevant T cell antigen.
  • An adjuvant may be used for supporting the immunization.
  • the immunization path may be selected from several useful immunization paths.
  • Preimmunization takes place under circumstances where the humoral immune response to the subsequently employed carrier molecule (antibody formation) is as low as pos ⁇ sible, while inducing T cell immunity to the subsequently employed carrier molecule.
  • the immunization takes place with the B cell anti ⁇ gen coupled on the carrier molecule (the T cell antigen in modified form compared with the preimmunization).
  • the immunization typically comprises the use of an adjuvant as an immune stimulating means, such as e.g. aluminium hydro- xide, Freund's adjuvant (complete or incomplete), saponin or another known adjuvant.
  • the type of B cell antigen may be any molecular configu ⁇ ration to which antibodies can be produced, e.g. a hapten, a peptide of any size, a carbohydrate, a lipid, a nucleic acid molecule, a protein having low immunogenecity or a combination of these.
  • the coupling chemistry should be well-defined as far as possible, i.e. the reactions taking place must be under ⁇ standable in a molecular sense, and they must be control- lable as follows:
  • B cell antigen is selectively coupled to the carrier protein, whereas formation of di- and polymeric aggregates is obviated, i.e. no or little formation of carrier- carrier or B cell antigen B cell antigen aggregates.
  • proteins When subjected to denaturing impacts, proteins tend to form large aggregates, primarily because of the establish ⁇ ment of intermolecular disulfide bridges, but also because of the establishment of non-covalent interactions between molecules.
  • This tendency can be counteracted/eliminated by various chemical methods, such as e.g. reduction of disulfide bridges (e.g. with dithiotreitol) followed by alkylation (e.g. with iodine acetamide) , or by modification of -C00H groups or -NH date groups. Then e.g. a denaturing heat treat- ment can be performed without causing precipitation.
  • various chemical methods such as e.g. reduction of disulfide bridges (e.g. with dithiotreitol) followed by alkylation (e.g. with iodine acetamide) , or by modification of -C00H groups or -NH date groups.
  • the solution is finally autoclaved for 1 hour at 105°C.
  • the solution is centrifuged (10,000 g) for 20 minutes, and the supernatant from this is dialysed against the desired buffer, following which the protein solution is sterile filtrated (0.22 ⁇ m) and is stored at 4°C in a suitable volume.
  • the protein concentration is determined by the method des ⁇ cribed by Bradford (Bradford, M.M. , Anal. Biochem. 22, 248, 1876).
  • mice Groups of mice (5 in each) were immunized with various doses of native antigen (ovalbumin), from 0.01 ⁇ g per immunization to 100 ⁇ g per immunization (fig. 3A) , or with various doses of denatured antigen (ovalbumin) (fig. 3B) .
  • the antigen was administered adsorbed to aluminium hydroxide, (A1(0H) confront).
  • the immunizations were repeated at intervals of 14 days, and blood samples were taken 10 days after each immunization for measurement of antibody amounts (titer).
  • the antibody amount (the titer) was measured by an ELISA method in which the antigen was applied to polystyrene plates.
  • Sera were diluted 1:1000, from which twofold di ⁇ lutions were performed. The titer can be evaluated on the basis of the serum dilution where the signal value (the ordinate) is 50% of the maximum signal value.
  • the figure shows the antibody response in the various groups after the 5th immunization. It will be seen that the threshold value for a positive response after immunization with native antigen is 0.01 ⁇ g/immuni- zation, whereas it is 0.5-1 ⁇ g/immunization after immuni ⁇ zation with denatured antigen.
  • the immunogenicity of native antigen is 50-100 times higher than that of denatured antigen.
  • N-hydroxysuccinimide activated hapten (9-thioguanine acetic acid) was coupled to heat denatured ovalbumin (OA) in a carbonate buffer, pH 8.0.
  • mice Groups of 5 mice each were then immunized with various doses of antigen, adsorped to A1(0H) overlook as adjuvant.
  • the mice were bled 10 days after each immunization (which took place at intervals of 14 days), and mouse sera were analyzed for antibody titer by an ELISA technique, partly to the hapten thioguanine, partly to the carrier, OA.
  • Figure 4 shows that there is a poor or low antibody titer to the carrier molecule at low doses of the antigen, and that at selected antigen doses it is only possible to obtain antibodies to the hapten.
  • the A-part of figure 5 shows the antibody response in mice, immunized with a hapten (merthiolate) coupled to native ovalbumin as a carrier molecule.
  • X shows the antibody response to the hapten, while o shows the antibody response to the carrier part.
  • 0.5 ⁇ g of antigen per immunization is used in the immunization.
  • the figure shows the antibody response after 3 immunizations.
  • the B-part of figure 5 shows the antibody response after immunization with hapten (merthiolate) coupled on de ⁇ natured antigen (ovalbumin).
  • x shows the antibody response to the hapten
  • o shows the antibody response to the carrier part.
  • 0.5 ⁇ g of antigen per immu ⁇ nization is used in the immunization.
  • the figure shows the antibody response after the 3rd immunization.
  • an immunization dose with denatured carrier protein where no antibody response is developed to the carrier part, but only to the hapten, can be selected from a dose response graph ( see examples 2 and 3).
  • mice with 5 mice in each group were immunized with a carrier molecule (denatured ovalbumin, 10 ⁇ g per immunization), to which various amounts of activated hap ⁇ ten (merthiolate) were coupled (indicated in figure 6 as the amount ( ⁇ l) of N-hydroxysuccinimide ester of merthio ⁇ late (40 mg/ml) in DMSO, added to 1 ml of denatured oval ⁇ bumin, 1 mg per ml in carbonate buffer, pH 8.0). After coupling the individual antigens were dialysed against PBS, and then they were adsorbed on adjuvant (A1(0H) protest) and used for the immunizations.
  • a carrier molecule denatured ovalbumin, 10 ⁇ g per immunization
  • activated hap ⁇ ten merthiolate
  • test comprised the use of from 0 to 100 ⁇ l of active merthiolate in the couplings.
  • the antibody response to hapten was analysed by an ELISA technique in which hapten (merthiolate) was used for the titration, coupled to an irrelevant carrier (diphtheria toxoid) for measurement of the hapten specific response, while uncoupled ovalbumin was used for the measurement of the antibody response to carrier (ovalbumin).
  • the antibody response is indicated in arbi ⁇ trary units, the antibody response to uncoupled carrier being put at 100 for the carrier reponse, while for the hapten response the maximum response after immunization with carrier, coupled with 100 ⁇ l of active methiolate, is put at 100 .
  • the selection of an immunization dose of carrier providing a significant antibody to uncoupled carrier and the increase in the hapten density result in a higher antibody response to hapten, while the anti carrier antibody response will be low or non-existing.
  • the peptide was synthetized manually on 1.5 g of an Fmoc- Arg(Mtr)-PepSyn KA resin using standard Fmoc strategy with either Pfp ester or BOP couplings.
  • the side chains were protected with t-butyl based groups with the exception of arginine which was protected with Mtr.
  • the peptide was separated from the solid carrier by treatment of 742 mg of the peptidyl resin with 93:2:2:2:1 TFA/H 2 0/thioanisole/ thiophenol/ethanedithiol for 16 hours. After filtration the peptide was isolated by reducing the volume from the TFA mixture in a nitrogen current and then precipitated with diethyl ether.
  • the precipitated peptide was redis- solved in 0.1% TFA and freeze dried, following which it was dissolved again in 0.1% TFA and purified by reverse phase chromatography.
  • the purified peptide eluted as a single peak by reverse phase HPLC and was further identi ⁇ fied by mass spectrometry: (M+H) 819.8.
  • KU834 was coupled to the carrier protein in five different peptide carrier ratios (mole:mole):
  • KU843 was photocoupled to the carrier protein in five dif ⁇ ferent peptide carrier ratios (mole:mole):
  • Peptide and carrier in 0.1 M NaHCO. were mixed in the de ⁇ sired coupling ratio, and the mixture was then illuminated for 30 minuttes in a photochemical Rayonet reactor (16 UV lamps, wavelength 350 nm).
  • mice were preimmunized with native carrier, or they received no preimmunization, and then they were immu- nized 14 days later with hapten coupled on native and de ⁇ natured carrier, respectively. 25 ⁇ g of carrier were used for all immunizations; see figure 7.
  • the example shows that when the same carrier conformation (here native) is used for preimmunization as well as for immunization, the antibody response to hapten (merthio ⁇ late) is suppressed compared with the situation where no preimmunization has taken place. If, on the other hand, a different carrier conformation is used for preimmunization and for the immunization with the hapten coupled composi ⁇ tion, the antibody response to hapten is increased com ⁇ pared with the situation where preimmunization has not taken place.
  • mice Groups of 5 mice each were preimmunized with 25 ⁇ g of na ⁇ tive carrier followed by varying time intervals ( 1 week to 12 months) of immunization with hapten coupled denatured carrier. Merthiolate was used as hapten and OA as carrier in the test. Immunization dose was 25 ⁇ g of hapten carrier complex .
  • Blood samples were taken 14 days after the 1st immuniza ⁇ tion with the hapten carrier complex, and these were ana- lyzed for antihapten antibody content.
  • the antihapten titer in the group which had been pre ⁇ immunized with native antigen 1 month before immunization with hapten carrier complex, has been given the value 100 arbitrary units.
  • Peritoneal macrophages were isolated from 20 CFlxBalb/c- mice. The macrophages were washed in a serum-free medium (DMEM), adjusted to a cell density of 2x10 ml and incubated (pulsed) for 20 minutes at 37°C with antigen. Three groups (A, B and C) were incubated with native ovalbumin (OA), 2 mg/ml, denatured OA, 2 mg/ml, and DMEM without antigen, respectively.
  • DMEM serum-free medium
  • mice group I received cells pulsed with native OA
  • mouse group II received cells pulsed with denatured OA
  • mouse group III received cells which had only been incubated with medium without antigen.
  • mice from each mouse group were immunized intraperitoneally with 100 ⁇ g native OA together with AI(0H) , a total of 0.5 ml, while the remaining 3 were immunized intraperitoneally with 100 ⁇ m denatured OA with AI(0H) 3 .
  • mice Three weeks later blood samples were taken from all the mice, and sera were analyzed for antibodies to denatured OA in the mice which had been ip-immunized with denatured OA, and to native OA in the mice which had been ip- immunized with native OA, respectively.
  • mice which have been given macrophages either pulsed with native or with denatured OA, will both develop T cell immunity capable of supporting immune response to both native and denatured OA.
  • This example confirms the assumption that native and denatured antigen both contain T cell epitopes capable of stimulating T accessory cells, and that these then subsequently can assist an antibody response to either the native or the denatured antigen.

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Abstract

Anticorps dirigés contre les haptènes et d'autres antigènes de lymphocyte B chez les animaux, et obtenus au moyen d'un procédé selon lequel on introduit dans un animal l'antigène de lymphocyte B sous une forme couplée à une molécule porteuse immunogène sous forme d'antigène protéique pouvant normalement induire l'immunité à la fois des lymphocytes T et des lymphocytes B. Le procédé est caractérisé en ce que l'antigène protéique immunogène est transformé en antigène de lymphocyte T exclusif ou prédominant par une modification par dénaturation de l'antigène, en ce que l'antigène de lymphocyte B contre lequel on veut diriger des anticorps est couplé à l'antigène de lymphocyte T transformé, à la suite de quoi on effectue l'immunisation éventuellement à l'aide d'un adjuvant servant de moyen immunostimulant, et de préférence après immunisation préalable au moyen d'un antigène de lymphocyte T sous une forme éventuellement différente de celle utilisée dans l'immunisation proprement dite avec l'antigène de lymphocyte B. Les antigènes ainsi produits sont utilisables notamment dans la production de vaccins à usage vétérinaire.
PCT/DK1993/000382 1992-11-24 1993-11-24 Procede de production d'anticorps avec des conjugues de l'antigene de lymphocytes b et une molecule porteuse denaturee Ceased WO1994012213A1 (fr)

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AU55610/94A AU5561094A (en) 1992-11-24 1993-11-24 Method of producing antibodies with conjugates of b-cell-antigen and denatured carrier

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DK140992A DK140992D0 (da) 1992-11-24 1992-11-24 Fremgangsmaade til frembringelse af antistoffer mod haptener og andre b-celle-antigener, antistoffer opnaaet ved fremgangsmaaden og anvendelse af disse antistoffer til fremstilling af vacciner, isaer til veterinaermedicinsk brug
DK1409/92 1992-11-24

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WO2001097842A1 (fr) * 2000-06-20 2001-12-27 Oleg Iliich Epshtein Procede de traitement d'un syndrome pathologique et produit medicamenteux
WO2002038611A3 (fr) * 2000-11-09 2003-01-09 Immunolex Lab Procede de production d'anticorps par immunisation avec des conjugues de molecules couplees a des proteines a charge modifiee
US7923009B2 (en) 2002-08-02 2011-04-12 Oleg Iliich Epshtein Medicinal agent and method for curing prostate diseases
US8066992B2 (en) 2002-08-02 2011-11-29 Oleg Iliich Epshtein Medicament and a method of treating a pathological syndrome
US8241625B2 (en) 2001-12-26 2012-08-14 Oleg Epshtein Medicament and a method for regulation of the vascular tone
US8637030B2 (en) 2010-07-15 2014-01-28 Oleg I. Epshtein Combination pharmaceutical composition and methods of treating functional diseases or conditions of gastrointestinal tract
US9308259B2 (en) 2006-06-06 2016-04-12 Oleg Iliich Epshtein Medicinal agent for treating fatness, diabetes, and diseases associated with impaired glucose tolerance
US9308275B2 (en) 2010-07-15 2016-04-12 Oleg Iliich Epshtein Method of increasing the effect of an activated-potentiated form of an antibody
US9561273B2 (en) 2010-07-15 2017-02-07 Oleg Iliich Epshtein Methods of treating multiple sclerosis

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0106285A2 (fr) * 1982-10-08 1984-04-25 Otsuka Pharmaceutical Co., Ltd. Antigène relatif à la liaison glycosidique, procédé de production et agent anticancéreux le contenant
WO1989006974A2 (fr) * 1988-02-01 1989-08-10 Praxis Biologics, Inc. Epitopes de cellules t a titre de molecules porteuses pour vaccins conjugues
EP0378881A1 (fr) * 1989-01-17 1990-07-25 ENIRICERCHE S.p.A. Peptides synthétiques et leur utilisation comme support universel pour la préparation de conjugués immunogènes convenant au développement de vaccins synthétiques
EP0427347A1 (fr) * 1989-11-10 1991-05-15 ENIRICERCHE S.p.A. Peptides synthétiques utiles comme porteurs universels pour la préparation des conjugués immunogéniques el leur emploi dans le développment des vaccins synthétiques
EP0429816A1 (fr) * 1989-10-31 1991-06-05 F. Hoffmann-La Roche Ag Composition de vaccin à base d'un épitope non-immunosuppressif de cellules T

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0106285A2 (fr) * 1982-10-08 1984-04-25 Otsuka Pharmaceutical Co., Ltd. Antigène relatif à la liaison glycosidique, procédé de production et agent anticancéreux le contenant
WO1989006974A2 (fr) * 1988-02-01 1989-08-10 Praxis Biologics, Inc. Epitopes de cellules t a titre de molecules porteuses pour vaccins conjugues
EP0378881A1 (fr) * 1989-01-17 1990-07-25 ENIRICERCHE S.p.A. Peptides synthétiques et leur utilisation comme support universel pour la préparation de conjugués immunogènes convenant au développement de vaccins synthétiques
EP0429816A1 (fr) * 1989-10-31 1991-06-05 F. Hoffmann-La Roche Ag Composition de vaccin à base d'un épitope non-immunosuppressif de cellules T
EP0427347A1 (fr) * 1989-11-10 1991-05-15 ENIRICERCHE S.p.A. Peptides synthétiques utiles comme porteurs universels pour la préparation des conjugués immunogéniques el leur emploi dans le développment des vaccins synthétiques

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DIALOG INFORMATION SERVICES, File 351, WPIL, Dialog Accession No. 009259776, TOSHIBA KK: "Method of Detecting Gene by Reacting Sample with Antibody to Single Stranded Oligo-Nucleotide - of Given Base Sequence, then Detecting Presence of Antigen-Antibody Reaction"; & JP,A,4 286 957, 12-10-1992, 9247, (Basic). *
METHODS IN ENZYMOLOGY, Volume 70, 1980, B. ERLANGER, "The Preparation of Antigenic Hapten-Carrier Conjugates: A Survey", page 85 - page 104. *
SCIENCE, Volume 249, July 1990, H.M. ETLINGER et al., "Use of Prior Vaccinations for the Development of New Vaccines", page 423 - page 425. *

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AU5561094A (en) 1994-06-22

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